Sucrose Assay Kit, BioAssay(TM)

Sucrose (C12H22O11) is a disaccharide of glucose and fructose with an a-1,2-glycosidic linkage. It is the most common food sweetener and the most important sugar in plants. In mammals, sucrose is readily digested in the stomach into glucose and fructose, which are rapidly absorbed into the bloodstream in the small intestine.\nSimple, direct and high-throughput assays for measuring sucrose concentrations find wide Applications:. Improved assay uses invertase to digest sucrose into fructose and glucose. The resulting fructose is then quantified using our fructose assay reagent. The measured color intensity at 565nm is directly proportional to the sucrose concentration in the sample.\n\nKey Features:\nNo interference by glucose. Use 20ul samples. Linear detection range: 17 to 2000uM sucrose.\n\nApplications:\nAssays: sucrose in biological samples (e.g. serum, plasma, urine, saliva, milk, culture medium), food, juice, beverage and other agricultural products.\nDrug Discovery/Pharmacology: effects of drugs on sucrose metabolism.\n\nKit Contents:\nAssay Buffer: 10ml Invertase: 120ul\nPMS Solution: 1.5ml Enzyme: 120ul\nMTT Solution: 1.5ml Standard: 400ul 40mM Sucrose\nStorage conditions. The kit is shipped on ice. Store all components at -20 degrees C. Shelf life of three months after receipt.\nPrecautions: reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.\n\nAssay Procedure:\nNote: (1) The following substances interfere and should be avoided in sample preparation: ascorbic acid, SDS (>0.2%), sodium azide, NP-40 (>1%) and Tween-20 (>1%). (2) This assay is based on a kinetic reaction. To ensure identical incubation time, addition of Working Reagent to standard and samples should be quick and mixing should be brief but thorough.\nSample treatment: liquid samples such as serum, plasma and fruit juices can be assayed directly. Milk samples should be cleared by mixing 600ul milk with 100ul 6 N HCl. Centrifuge 5 min at 14,000 rpm. Transfer 300ul supernatant into a clean tube and neutralize with 50ul 6 N NaOH. The neutralized supernatant is ready for assay (dilution factor n=1.36).\n1. Equilibrate all components to room temperature. Briefly centrifuge the tubes before opening. Keep thawed tubes on ice during assay.\n2. Standards: mix 12ul 40mM Standard with 228ul dH2O (final 2000uM). Dilute standard in dH2O as follows.\nNo 2000uM STD+H2O Vol (ul) Sucrose (uM)\n1 100ul+0ul 100 2000\n2 60ul+40ul 100 1200\n3 30ul+70ul 100 600\n4 0ul +100ul 100 0\nTransfer 20ul diluted standards into separate wells of a clear flatbottom 96-well plate. Samples: transfer 20ul of each sample into separate wells of the plate. Note: if a sample is known to contain fructose, prepare an extra sample blank well with 20ul of the sample.\n3. Color reaction. Prepare enough Working Reagent by mixing, for each reaction well, 56ul Assay Buffer, 1ul Invertase, 1ul Enzyme, 14ul PMS Solution and 14ul MTT Solution. Add 80ul Working Reagent to each well.\nNote: for the sample that contains fructose, prepare a blank control reagent with no Invertase (i.e., 56ul Assay Buffer, 1ul Enzyme, 14ul PMS Solution and 14ul MTT Solution). Add 80ul of the control Reagent to each Sample Blank well. Immediately tap plate to mix. Incubate 60 min at room temperature.\n4. Read optical density at 565nm (520-600nm).\nNote: If the calculated sucrose concentration of a sample is higher than 2000uM, dilute sample in water and repeat the assay. Multiply result by the dilution factor n.\n\nCalculation:\nSubtract blank value (water, #4) from the standard values and plot the DOD against standard concentrations. Determine the slope and calculate the sucrose concentration of Sample,\n[Sucrose] =\nODSAMPLE

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SPECIFICATIONS

Catalog Number

S8010-21

Size

1Kit

References

1. Harrison, C.S. and Faler, K.T. (1957). Analysis of the chemical sucrose determination of extracellular fluid volume using C14-labeled sucrose. Am. J. Physiol. 188: 568-570.\n2. Holmes, E.W. (1997). Coupled enzymatic assay for the determination of sucrose. Anal. Biochem. 244: 103-109.\n3. Young, M.K. and Prudden, J.F. (1954). Simultaneous determination of sucrose and inulin in biologic fluids. J. Lab. Clin. Med. 44: 160-165.